Phenylalanyl-tRNA synthetase from Staphylococcus aureus

ABSTRACT

The invention provides tRNA synthetase polypeptides and DNA (RNA) encoding tRNA synthetase polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing tRNA synthetase polypeptide for the protection against infection, particularly baterial infections.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, in these and inother regards, the invention relates to novel polynucleotides andpolypeptides of the tRNA synthetase family, hereinafter referred to as"tRNA synthetase".

BACKGROUND OF THE INVENTION

It is particularly preferred to employ Staphylococcal genes and geneproducts as targets for the development of antibiotics. TheStaphylococci make up a medically important genera of microbes. They areknown to produce two types of disease, invasive and toxigenic. Invasiveinfections are characterized generally by abscess formation effectingboth skin surfaces and deep tissues. S. aureus is the second leadingcause of bacteremia in cancer patients. Osteomyelitis, septic arthritis,septic thrombophlebitis and acute bacterial endocarditis are alsorelatively common. There are at least three clinical conditionsresulting from the toxigenic properties of Staphylococci. Themanifestation of these diseases result from the actions of exotoxins asopposed to tissue invasion and bacteremia. These conditions include:Staphylococcal food poisoning, scalded skin syndrome and toxic shocksyndrome.

The tRNA synthetases have a primary role in protein synthesis accordingto the following scheme: ##EQU1## in which AA is an amino acid.

Inhibition of this process leads to a reduction in the levels of chargedtRNA and this triggers a cascade of responses known as the stringentresponse, the result of which is the induction of a state of dormancy inthe organism. As such selective inhibitors of bacterial tRNA synthetasehave potential as antibacterial agents. One example of such is mupirocinwhich is a selective inhibitor of isoleucyl tRNA synthetase. Isolationof tRNA synthetase allows for the identification and analysis ofpotential antibacterial targets to facilitate screening forantibacterial compounds.

Isoleucyl tRNA synthetase, isolated from Staphylococcus aureus, hasalready been described (Chalker, A., F., Ward, J., M., Fosberry, A., P.and Hodgson, J., E. 1994 Gene 141: 103-108).

Clearly, there is a need for factors that may be used to screencompounds for antibiotic activity and which factors may also be used todetermine their roles in pathogenesis of infection, dysfunction anddisease. There is a need, therefore, for identification andcharacterization of such factors and their antagonists and agonistswhich can play a role in preventing, ameliorating or correctinginfections, dysfunctions or diseases.

The polypeptides of the invention have amino acid sequence homology to aknown phenylalanyl tRNA synthetase protein.

SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have beenidentified as novel tRNA synthetase polypeptides by homology between theamino acid sequence set out in FIGS. 2 or 4 SEQ ID NO: 2 and 4! and aknown amino acid sequence or sequences of other proteins such asphenylalanyl tRNA synthetase protein.

It is a further object of the invention to provide polynucleotides thatencode tRNA synthetase polypeptides, particularly polynucleotides thatencode the polypeptide herein designated tRNA synthetase.

In a particularly preferred embodiment of this aspect of the inventionthe polynucleotide comprises a region encoding phenylalanyl tRNAsynthetase polypeptides comprising the sequence set out in FIG. 1 SEQ IDNO: 1! or FIG. 3 SEQ ID NO: 3!, or variant of either sequence.

In another particularly preferred embodiment of the invention there is anovel phenylalanyl tRNA synthetase protein from Staphylococcus aureuscomprising the amino acid sequence of FIG. 2 SEQ ID NO: 2! or FIG. 4 SEQID NO: 4!, or a variant of either sequence.

In accordance with this aspect of the invention there is provided anisolated nucleic acid molecule encoding a mature polypeptide expressibleby the Staphylococcus aureus WCUH 29 strain contained in NCIMB DepositNo. 40771.

In accordance with this aspect of the invention there are providedisolated nucleic acid molecules encoding tRNA synthetase, particularlyStaphylococcus aureus tRNA synthetase, including mRNAs, cDNAs, genomicDNAs. Further embodiments of this aspect of the invention includebiologically, diagnostically, prophylactically, clinically ortherapeutically useful variants thereof, and compositions comprising thesame.

In accordance with another aspect of the invention, there is providedthe use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of this aspect of the invention arenaturally occurring allelic variants of tRNA synthetase and polypeptidesencoded thereby.

In accordance with this aspect of the invention there are provided novelpolypeptides of Staphylococcus aureus referred to herein as tRNAsynthetase as well as biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

Among the particularly preferred embodiments of this aspect of theinvention are variants of tRNA synthetase polypeptide encoded bynaturally occurring alleles of the tRNA synthetase gene.

In a preferred embodiment of this aspect of the invention there areprovided methods for producing the aforementioned tRNA synthetasepolypeptides.

In accordance with yet another aspect of the invention, there areprovided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

In accordance with certain preferred embodiments of this aspect of theinvention, there are provided products, compositions and methods for (i)assessing tRNA synthetase expression, (ii) treating disease, forexample, disease, such as, infections of the upper respiratory tract(e.g., otitis media, bacterial tracheitis, acute epiglottitis,thyroiditis), lower respiratory (e.g., empyema, lung abscess), cardiac(e.g., infective endocarditis), gastrointestinal (e.g., secretorydiarrhoea, splenic absces, retroperitoneal abscess), CNS (e.g., cerebralabscess), eye (e.g., blepharitis, conjunctivitis, keratitis,endophthalmitis, preseptal and orbital cellulitis, darcryocystitis),kidney and urinary tract (e.g., epididymitis, intrarenal and perinephricabsces, toxic shock syndrome), skin (e.g., impetigo, folliculitis,cutaneous abscesses, cellulitis, wound infection, bacterial myositis)bone and joint (e.g., septic arthritis, osteomyelitis), (iii) assayinggenetic variation, (iv) and administering a tRNA synthetase polypeptideor polynucleotide to an organism to raise an immunological responseagainst a bacteria, especially a Staphylococcus aureus bacteria.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided polynucleotides thathybridize to tRNA synthetase polynucleotide sequences, particularlyunder stringent conditions.

In certain preferred embodiments of this aspect of the invention thereare provided antibodies against tRNA synthetase polypeptides.

In accordance with another aspect of the invention, there are providedtRNA synthetase agonists and antagonists each of which are alsopreferably bacteriostatic or bacteriocidal.

In a further aspect of the invention there are provided compositionscomprising a tRNA synthetase polynucleotide or a tRNA synthetasepolypeptide for administration to a cell or to a multicellular organism.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings depict certain embodiments of the invention. Theyare illustrative only and do not limit the invention otherwise disclosedherein.

FIGS. 1 through 1B show the polynucleotide sequence of Staphylococcusaureus phenylalanyl tRNA synthetase alpha subunit SEQ ID NO: 1!.

FIGS. 2 through shows the amino acid sequence of Staphylococcus aureusphenylalanyl tRNA synthetase alpha subunit SEQ ID NO: 2! deduced fromthe polynucleotide sequence of FIG. 1.

FIGS. 3 through 3D show the polynucleotide sequence of Staphylococcusaureus phenylalanyl tRNA synthetase beta subunit SEQ ID NO: 3!.

FIGS. 4 through 4A show the amino acid sequence of Staphylococcus aureusphenylalanyl tRNA synthetase beta subunit SEQ ID NO: 4! deduced from thepolynucleotide sequence of FIG. 3.

GLOSSARY

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

"Host cell" is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

"Identity," as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, "identity" also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. "Identity" and "similarity" can be readilycalculated by known methods (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991). While there exist a number of methodsto measure identity and similarity between two sequences, both terms arewell known to skilled artisans (Sequence Analysis in Molecular Biology,von Heinje, G., Academic Press, 1987; Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988).Methods commonly employed to determine identity or similarity betweensequences include, but are not limited to those disclosed in Carillo,H., and Lipman, D., SIAM J. Applied Math, 48: 1073 (1988). Preferredmethods to determine identity are designed to give the largest matchbetween the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include, but are not limited to, GCG programpackage (Devereux, J., et al., Nucleic Acids Research 12(1): 387(1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990)). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990)).

"Isolated" means altered "by the hand of man" from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, polynucleotide or apolypeptide naturally present in a living organism is not "isolated,"but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is "isolated", as the term is employedherein.

"Polynucleotide(s)" generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. "Polynucleotide(s)" include, without limitation, single-anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded, or a mixture of single- and double-stranded regions. Inaddition, polynucleotide as used herein refers to triple-strandedregions comprising RNA or DNA or both RNA and DNA. The strands in suchregions may be from the same molecule or from different molecules. Theregions may include all of one or more of the molecules, but moretypically involve only a region of some of the molecules. One of themolecules of a triple-helical region often is an oligonucleotide. Asused herein, the term "polynucleotide(s)" includes DNAs or RNAs asdescribed above that contain one or more modified bases. Thus, DNAs orRNAs with backbones modified for stability or for other reasons are"polynucleotide(s)" as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term"polynucleotide(s)" as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells, inter alia."Polynucleotide(s)" embraces short polynucleotides often referred to asoligonucleotide(s).

"Polypeptide(s)" refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. "Polypeptide(s)" refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may contain amino acidsother than the 20 gene encoded amino acids. "Polypeptide(s)" includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques which are well known to the art. Such modifications are welldescribed in basic texts and in more detailed monographs, as well as ina voluminous research literature, and they are well known to those ofskill in the art. It will be appreciated that the same type ofmodification may be present in the same or varying degree at severalsites in a given polypeptide. Also, a given polypeptide may contain manytypes of modifications. Modifications can occur anywhere in apolypeptide, including the peptide backbone, the amino acid side-chainsand the amino or carboxyl termini. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, glycosylation, lipid attachment, sulfation,gamma-carboxylation of glutamic acid residues, hydroxylation andADP-ribosylation, selenoylation, sulfation, transfer-RNA mediatedaddition of amino acids to proteins such as arginylation, andubiquitination. See, for instance, PROTEINS--STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork (1993) and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York(1983); Seifter et al., Meth Enzymol. 182: 626-646 (1990) and Rattan etal., Protein Synthesis: Posttranslational Modifications and Aging, Ann.N.Y. Acad. Sci. 663: 48-62 (1992). Polypeptides may be branched orcyclic, with or without branching. Cyclic, branched and branchedcircular polypeptides may result from post-translational naturalprocesses and may be made by entirely synthetic methods, as well.

"Variant(s)" as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods know to skilledartisans.

DESCRIPTION OF THE INVENTION

The invention relates to novel tRNA synthetase polypeptides andpolynucleotides as described in greater detail below. In particular, theinvention relates to polypeptides and polynucleotides of a novel tRNAsynthetase gene of Staphylococcus aureus, which is related by amino acidsequence homology to phenylalanyl tRNA synthetase polypeptide. Theinvention relates especially to tRNA synthetase having the nucleotideand amino acid sequences set out in FIGS. 1, 2, 3 and 4 SEQ ID NO: 1-4!,and to the tRNA synthetase nucleotide sequences of the DNA in NCIMBDeposit No. 40771 and amino acid sequences encoded thereby.

Techniques are available to evaluate temporal gene expression inbacteria, particularly as it applies to viability under laboratory andhost infection conditions. A number of methods can be used to identifygenes which are essential to survival per se, or essential to theestablishment and/or maintenance of an infection. Identification ofexpression of a sequence by one of these methods yields additionalinformation about its function and assists in the selection of suchsequence for further development as a screening target. Briefly, theseapproaches include, for example:

1) Signature Tagged Mutagenesis (STM)

This technique is described by Hensel et al., Science 269:400-403(1995), the contents of which is incorporated by reference forbackground purposes. Signature tagged mutagenesis identifies genesnecessary for the establishment/maintenance of infection in a giveninfection model.

The basis of the technique is the random mutagenesis of target organismby various means (e.g., transposons) such that unique DNA sequence tagsare inserted in close proximity to the site of mutation. The tags from amixed population of bacterial mutants and bacteria recovered from aninfected hosts are detected by amplification, radiolabeling andhybridization analysis. Mutants attenuated in virulence are revealed byabsence of the tag from the pool of bacteria recovered from infectedhosts.

In Staphylococcus aureus, because the transposon system is less welldeveloped, a more efficient way of creating the tagged mutants is to usethe insertion-duplication mutagenesis technique as described by Morrisonet al., J. Bacteriol. 159: 870 (1984) the contents of which isincorporated by reference for background purposes.

2) In Vivo Expression Technology (IVET)

This technique is described by Camilli et al., Proc. Nat'l. Acad. Sci.USA. 91: 2634-2638 (1994) and Mahan et al., Infectious Agents andDiseases 2: 263-268 (1994), the contents of each of which isincorporated by reference for background purposes. IVET identifies genesup-regulated during infection when compared to laboratory cultivation,implying an important role in infection. Sequences identified by thistechnique are implied to have a significant role in infectionestablishment/maintenance.

In this technique random chromosomal fragments of target organism arecloned upstream of a promoter-less reporter gene in a plasmid vector.The pool is introduced into a host and at various times after infectionbacteria may be recovered and assessed for the presence of reporter geneexpression. The chromosomal fragment carried upstream of an expressedreporter gene should carry a promoter or portion of a gene normallyupregulated during infection. Sequencing upstream of the reporter geneallows identification of the up regulated gene.

3) Differential display

This technique is described by Chuang et al., J Bacteriol. 175:2026-2036 (1993), the contents of which is incorporated by reference forbackground purposes. This method identifies those genes which areexpressed in an organism by identifying mRNA present usingrandomly-primed RT-PCR. By comparing pre-infection and post infectionprofiles, genes up and down regulated during infection can be identifiedand the RT-PCR product sequenced and matched to library sequences.

4) Generation of conditional lethal mutants by transposon mutagenesis.

This technique, described by de Lorenzo, V. et al., Gene 123: 17-24(1993); Neuwald, A. F. et al., Gene 125: 69-73(1993); and Takiff, H. E.et al., J. Bacteriol. 174: 1544-1553(1992), the contents of which isincorporated by reference for background purposes, identifies geneswhose expression are essential for cell viability.

In this technique transposons carrying controllable promoters, whichprovide transcription outward from the transposon in one or bothdirections, are generated. Random insertion of these transposons intotarget organisms and subsequent isolation of insertion mutants in thepresence of inducer of promoter activity ensures that insertions whichseparate promoter from coding region of a gene whose expression isessential for cell viability will be recovered. Subsequent replicaplating in the absence of inducer identifies such insertions, since theyfail to survive. Sequencing of the flanking regions of the transposonallows identification of site of insertion and identification of thegene disrupted. Close monitoring of the changes in cellularprocesses/morphology during growth in the absence of inducer yieldsinformation on likely function of the gene. Such monitoring couldinclude flow cytometry (cell division, lysis, redox potential, DNAreplication), incorporation of radiochemically labeled precursors intoDNA, RNA, protein, lipid, peptidoglycan, monitoring reporter enzyme genefusions which respond to known cellular stresses.

5) Generation of conditional lethal mutants by chemical mutagenesis.

This technique is described by Beckwith, J., Methods in Enzymology 204:3-18(1991), the contents of which are incorporated herein by referencefor background purposes. In this technique random chemical mutagenesisof target organism, growth at temperature other than physiologicaltemperature (permissive temperature) and subsequent replica plating andgrowth at different temperature (e.g., 42° C. to identify ts, 25° C. toidentify cs) are used to identify those isolates which now fail to grow(conditional mutants). As above close monitoring of the changes upongrowth at the non-permissive temperature yields information on thefunction of the mutated gene. Complementation of conditional lethalmutation by library from target organism and sequencing of complementinggene allows matching with library sequences.

Each of these techniques may have advantages or disadvantage dependingon the particular application. The skilled artisan would choose theapproach that is the most relevant with the particular end use in mind.For example, some genes might be recognised as essential for infectionbut in reality are only necessary for the initiation of infection and sotheir products would represent relatively unattractive targets forantibacterials developed to cure established and chronic infections.

6) RT-PCR

Bacterial messenger RNA, preferably that of Staphylococcus aureus, isisolated from bacterial infected tissue, e.g., 48 hour murine lunginfections, and the amount of each mRNA species assessed by reversetranscription of the RNA sample primed with random hexanucleotidesfollowed by PCR with gene specific primer pairs. The determination ofthe presence and amount of a particular mRNA species by quantificationof the resultant PCR product provides information on the bacterial geneswhich are transcribed in the infected tissue. Analysis of genetranscription can be carried out at different times of infection to gaina detailed knowledge of gene regulation in bacterial pathogenesisallowing for a clearer understanding of which gene products representtargets for screens for novel antibacterials. Because of the genespecific nature of the PCR primers employed it should be understood thatthe bacterial mRNA preparation need not be free of mammalian RNA. Thisallows the investigator to carry out a simple and quick RNA preparationfrom infected tissue to obtain bacterial mRNA species which are veryshort lived in the bacterium (in the order of 2 minute halflives).Optimally the bacterial mRNA is prepared from infected murine lungtissue by mechanical disruption in the presence of TRIzole (GIBCO-BRL)for very short periods of time, subsequent processing according to themanufacturers of TRIzole reagent and DNAase treatment to removecontaminating DNA. Preferably the process is optimized by finding thoseconditions which give a maximum amount of bacterial 16S ribosomal RNA,preferably that of Staphylococcus aureus, as detected by probingNortherns with a suitably labeled sequence specific oligonucleotideprobe. Typically, a 5' dye labelled primer is used in each PCR primerpair in a PCR reaction which is terminated optimally between 8 and 25cycles. The PCR products are separated on 6% polyacrylamide gels withdetection and quantification using GeneScanner (manufactured by ABI).

Use of the of these technologies when applied to the sequences of theinvention enables identification of bacterial proteins expressed duringinfection, inhibitors of which would have utility in anti-bacterialtherapy.

Deposited materials

A deposit containing a Staphylococcus aureus WCUH 29 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (NCIMB), 23 St. Machar Drive, Aberdeen AB2 1RY, Scotlandon 11 Sep. 1995 and assigned NCIMB Deposit No. 40771. The Staphylococcusaureus strain deposit is referred to herein as "the deposited strain" oras "the DNA of the deposited strain."

The deposited material is a strain that contains the full length tRNAsynthetase DNA, referred to as "NCIMB 40771" upon deposit.

The sequence of the polynucleotides contained in the deposited material,as well as the amino acid sequence of the polypeptide encoded thereby,are controlling in the event of any conflict with any description ofsequences herein.

The deposit has been made under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Micro-organisms for Purposesof Patent Procedure. The strain will be irrevocably and withoutrestriction or condition released to the public upon the issuance of apatent. The deposit is provided merely as convenience to those of skillin the art and is not an admission that a deposit is required forenablement, such as that required under 35 U.S.C. §112.

A license may be required to make, use or sell the deposited materialsand no such license is hereby granted.

Polypeptides

The polypeptides of the invention include the alpha subunit polypeptideof FIG. 2 SEQ ID NO: 2! and the beta subunit polypeptide of FIG. 4 SEQID NO: 4! (in particular the mature polypeptide) as well as polypeptidesand fragments, particularly those which have the biological activity oftRNA synthetase or a subunit thereof, and also those which have at least70% identity to the polypeptide of FIG. 2 SEQ ID NO: 2! or FIG. 4 SEQ IDNO: 4! or the relevant portion of either, preferably at least 80%identity to the polypeptide of FIG. 2 SEQ ID NO: 2! or FIG. 4 SEQ ID NO:4!, and more preferably at least 90% similarity (more preferably atleast 90% identity) to the polypeptide of FIG. 2 SEQ ID NO: 2! or FIG. 4SEQ ID NO: 4! and still more preferably at least 95% similarity (stillmore preferably at least 95% identity) to the polypeptide of FIG. 2 SEQID NO: 2! or FIG. 4 SEQ ID NO: 4! and also include portions of suchpolypeptides with such portion of the polypeptide generally containingat least 30 amino acids and more preferably at least 50 amino acids.

Variants that are fragments of the polypeptides of the invention may beemployed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, these variants may be employed asintermediates for producing the full-length polypeptides. Variants thatare fragments of the polynucleotides of the invention may be used tosynthesize full-length polynucleotides of the invention.

A fragment is a variant polypeptide having an amino acid sequence thatentirely is the same as part but not all of the amino acid sequence ofthe aforementioned polypeptides. As with tRNA synthetase polypeptidesfragments may be "free-standing," or comprised within a largerpolypeptide of which they form a part or region, most preferably as asingle continuous region, a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of the amino acid sequence of FIG. 2 SEQ ID NO: 2! or FIG. 4SEQ ID NO: 4!, or of variants of either sequence, except for deletion ofa continuous series of residues that includes the amino terminus, or acontinuous series of residues that includes the carboxyl terminus ordeletion of two continuous series of residues, one including the aminoterminus and one including the carboxyl terminus. Degradation forms ofthe polypeptides of the invention in a host cell, particularly aStaphylococcus aureus, are also preferred. Also preferred are fragmentscharacterized by structural or functional attributes such as fragmentsthat comprise alpha-helix and alpha-helix forming regions, beta-sheetand beta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions.

Also preferred are biologically active fragments which fragments arethose fragments that mediate activities of tRNA synthetase, includingthose fragments with a similar activity or an improved activity, or witha decreased undesirable activity. Also included are those that areantigenic or immunogenic in an animal, especially in a human.

Polynucleotides

Another aspect of the invention relates to isolated polynucleotideswhich encode the tRNA synthetase polypeptide having the deduced aminoacid sequence of FIG. 2 SEQ ID NO: 2! or FIG. 4 SEQ ID NO: 4! andpolynucleotides closely related to either sequence.

Using the information provided herein, such as the polynucleotidesequence set out in FIG. 1 SEQ ID NO: 1! or FIG. 3 SEQ ID NO: 3!, apolynucleotide of the invention encoding tRNA synthetase polypeptide maybe obtained using standard cloning and screening, such as those forcloning and sequencing chromosomal DNA fragments from Staphylococcusaureus WCUH 29 cells as starting material, followed by obtaining a fulllength clone. For example, to obtain a polynucleotide sequence of theinvention, such as that sequence given in FIG. 1 SEQ ID NO: 1!,typically a library of clones of chromosomal DNA of Staphylococcusaureus WCUH 29 in E. coli or some other suitable host is probed with aradiolabeled oligonucleotide, preferably a 17-mer or longer, derivedfrom a partial sequence. Clones carrying DNA identical to that of theprobe can then be distinguished using stringent conditions. Bysequencing the individual clones thus identified with sequencing primersdesigned from the original sequence it is then possible to extend thesequence in both directions to determine the full gene sequence.Conveniently such sequencing is performed using denatured doublestranded DNA prepared from a plasmid clone. Suitable techniques aredescribed by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989). (see Screening By Hybridization1.90 and Sequencing Denatured Double-Stranded DNA Templates 13.70).Illustrative of the invention, the polynucleotide set out in FIG. 1 SEQID NO: 1! and FIG. 3 SEQ ID NO: 3! was discovered in a DNA libraryderived from Staphylococcus aureus WCUH 29.

The DNA sequences thus obtained is set out in FIG. 1 SEQ ID NO: 1! andFIG. 3 SEQ ID NO: 3!. Each contains an open reading frame encoding a fora tRNA synthetase subunit protein having about the number of amino acidresidues set forth in FIG. 2 SEQ ID NO: 2! and FIG. 4 SEQ ID NO: 4!respectively with a deduced molecular weight that can be calculatedusing amino acid residue molecular weight values well known in the art.tRNA synthetase of the invention is structurally related to otherproteins of the tRNA synthetase family, as shown by the results ofsequencing the DNA encoding tRNA synthetase of the deposited strain. Theprotein exhibits greatest homology to phenylalanyl tRNA synthetaseprotein among known proteins. Phenylalanyl tRNA synthetase alpha subunitof FIG. 2 SEQ ID NO: 2! has about 64% identity over its entire lengthand about 77% similarity over its entire length with the amino acidsequence of Bacillus subtilis phenylalanyl tRNA synthetase alpha subunitpolypeptide. Phenylalanyl tRNA synthetase beta subunit of FIG. 4 SEQ IDNO: 4! has about 49% identity over its entire length and about 68%similarity over its entire length with the amino acid sequence ofBacillus subtilis phenylalanyl tRNA beta subunit synthetase polypeptide.

Sequence of the invention may also be identical over its entire lengthto the coding sequence in FIG. 1 SEQ ID NO: 1! or FIG. 3 SEQ ID NO: 3!.

Also provide by the invention is the coding sequence for the maturepolypeptide or a fragment thereof, by itself as well as the codingsequence for the mature polypeptide or a fragment in reading frame withother coding sequence, such as those encoding a leader or secretorysequence, a pre-, or pro- or prepro- protein sequence. Thepolynucleotide may also contain non-coding sequences, including forexample, but not limited to non-coding 5' and 3' sequences, such as thetranscribed, non-translated sequences, termination signals, ribosomebinding sites, sequences that stabilize mRNA, introns, polyadenylationsignals, and additional coding sequence which encode additional aminoacids. For example, a marker sequence that facilitates purification ofthe fused polypeptide can be encoded. In certain embodiments of thisaspect of the invention, the marker sequence is a hexa-histidinepeptide, as provided in the pQE vector (Qiagen, Inc.) and described inGentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824 (1989), or an HAtag (Wilson et al., Cell 37: 767 (1984)). Polynucleotides of theinvention also include, but are not limited to, polynucleotidescomprising a structural gene and its naturally associated sequences thatcontrol gene expression.

In accordance with the foregoing, the term "polynucleotide encoding apolypeptide" as used herein encompasses polynucleotides which include asequence encoding a polypeptide of the invention, particularlybacterial, and more particularly the Staphylococcus aureus tRNAsynthetase having the amino acid sequence set out in FIG. 2 SEQ ID NO:2!. The term encompasses polynucleotides that include a singlecontinuous region or discontinuous regions encoding the polypeptide (forexample, interrupted by integrated phage or insertion sequence orediting) together with additional regions, that also may contain codingand/or non-coding sequences.

The invention further relates to variants of the herein above describedpolynucleotides which encode for variants of the polypeptide having thededuced amino acid sequence of FIG. 2 SEQ ID NO: 2! or FIG. 4 SEQ ID NO:4!.

Further particularly preferred embodiments are polynucleotides encodingtRNA synthetase variants, which have the amino acid sequence of tRNAsynthetase polypeptide of FIG. 2 SEQ ID NO: 2! or FIG. 4 SEQ ID NO: 4!in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acidresidues are substituted, deleted or added, in any combination.Especially preferred among these are silent substitutions, additions anddeletions, which do not alter the properties and activities of tRNAsynthetase.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideencoding tRNA synthetase polypeptide having the amino acid sequence setout in FIG. 2 SEQ ID NO: 2! or FIG. 4 SEQ ID NO: 4!, and polynucleotideswhich are complementary to such polynucleotides. Alternatively, mosthighly preferred are polynucleotides that comprise a region that is atleast 80% identical over their entire length to a polynucleotideencoding tRNA synthetase polypeptide of the Staphylococcus aureus DNA ofthe deposited strain and polynucleotides complementary thereto. In thisregard, polynucleotides at least 90% identical over their entire lengthto the same are particularly preferred, and among these particularlypreferred polynucleotides, those with at least 95% are especiallypreferred. Furthermore, those with at least 97% are highly preferredamong those with at least 95%, and among these those with at least 98%and at least 99% are particularly highly preferred, with at least 99%being the more preferred.

Preferred embodiments in this respect, moreover, are polynucleotideswhich encode polypeptides which retain substantially the same biologicalfunction or activity as the mature polypeptide encoded by the DNA ofFIG. 1 SEQ ID NO: 1! or FIG. 3 SEQ ID NO: 3!.

The invention further relates to polynucleotides that hybridize to theherein above-described sequences. In this regard, the inventionespecially relates to polynucleotides which hybridize under stringentconditions to the herein above-described polynucleotides. As hereinused, the terms "stringent conditions" and "stringent hybridizationconditions" mean hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences. An exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the filters in 0.1×SSC at about65° C. Hybridization and wash conditions are well known and exemplifiedin Sambrook, et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor, N.Y., (1989), particularly Chapter 11therein, the disclosure of which is hereby incorporated in its entiretyby reference.

The invention also provides a polynucleotide consisting essentially of apolynucleotide sequence obtainable by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set forth inSEQ ID NO: 1 or SEQ ID NO: 3 under stringent hybridization conditionswith a probe having the sequence of said polynucleotide sequence setforth in SEQ ID NO: 1 or SEQ ID NO: 3 or a fragment of either sequence;and isolating said DNA sequence. Fragments useful for obtaining such apolynucleotide include, for example, probes and primers describedelsewhere herein.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for RNA, cDNA and genomicDNA to isolate full-length cDNAs and genomic clones encoding tRNAsynthetase and to isolate cDNA and genomic clones of other genes thathave a high sequence similarity to the tRNA synthetase gene. Such probesgenerally will comprise at least 15 bases. Preferably, such probes willhave at least 30 bases and may have at least 50 bases. Particularlypreferred probes will have at least 30 bases and will have 50 bases orless.

For example, the coding region of the tRNA synthetase gene may beisolated by screening using the known DNA sequence to synthesize anoligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a gene of the invention is then used to screena library of cDNA, genomic DNA or mRNA to determine which members of thelibrary the probe hybridizes to.

The polynucleotides and polypeptides of the invention may be employed asresearch reagents and materials for discovery of treatments of anddiagnostics for disease, particularly human disease, as furtherdiscussed herein relating to polynucleotide assays, inter alia.

The polynucleotides of the invention that are oligonucleotides derivedfrom the sequences of SEQ ID NOS: 1, 2, 3, or 4 may be used in theprocesses herein as described, but preferably for PCR, to determinewhether or not the ploynucleotides identified herein in whole or in partare transcribed in infected tissue. It is recognized that such sequenceswill also have utility in diagnosis of the stage of infection and typeof infection the pathogen has attained.

The polynucleotides may encode a polypeptide which is the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature polypeptide (when the mature form has more thanone polypeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, may allowprotein transport, may lengthen or shorten protein half-life or mayfacilitate manipulation of a protein for assay or production, amongother things. As generally is the case in vivo, the additional aminoacids may be processed away from the mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences which are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Vectors, host cells, expression

The invention also relates to vectors which comprise a polynucleotide orpolynucleotides of the invention, host cells which are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof or polynucleotides ofthe invention. Introduction of a polynucleotide into the host cell canbe effected by methods described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), suchas, calcium phosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, E. coli, streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanomacells; and plant cells.

A great variety of expression systems can be used to produce apolypeptide of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic Assays

This invention is also related to the use of the tRNA synthetasepolynucleotides of the invention for use as diagnostic reagents.Detection of tRNA synthetase in a eukaryote, particularly a mammal, andespecially a human, will provide a diagnostic method for diagnosis of adisease. Eukaryotes (herein also "individual(s)"), particularly mammals,and especially humans, infected with an organism comprising the tRNAsynthetase gene may be detected at the DNA level by a variety oftechniques.

Nucleic acids for diagnosis may be obtained from an infectedindividual's cells and tissues, such as bone, blood, muscle, cartilage,and skin. Genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniqueprior to analysis. RNA or cDNA may also be used in the same ways. Usingamplification, characterization of the strain of prokaryote present in aeukaryote, particularly a mammal, and especially a human, may be made byan analysis of the genotype of the prokaryote gene. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to the genotype of a reference sequence. Point mutationscan be identified by hybridizing amplified DNA to labeled tRNAsynthetase polynucleotide sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase digestion or bydifferences in melting temperatures. DNA sequence differences may alsobe detected by alterations in the electrophoretic mobility of the DNAfragments in gels, with or without denaturing agents, or by direct DNAsequencing. See, e.g. Myers et al., Science, 230: 1242 (1985). Sequencechanges at specific locations also may be revealed by nucleaseprotection assays, such as RNase and S1 protection or a chemicalcleavage method. See, e.g., Cotton et al., Proc. Natl. Acad. Sci., USA,85: 4397-4401 (1985).

Cells carrying mutations or polymorphisms in the gene of the inventionmay also be detected at the DNA level by a variety of techniques, toallow for serotyping, for example. For example, RT-PCR can be used todetect mutations. It is particularly preferred to used RT-PCR inconjunction with automated detection systems, such as, for example,GeneScan. RNA or cDNA may also be used for the same purpose, PCR orRT-PCR. As an example, PCR primers complementary to the nucleic acidencoding tRNA synthetase can be used to identify and analyze mutations.These primers may be used for amplifying tRNA synthetase DNA isolatedfrom a sample derived from an individual. The invention also providesthese primers with 1, 2, 3 or 4 nucleotides removed from the 5' and/orthe 3' end. The primers may be used to amplify the gene isolated from aninfected individual such that the gene may then be subject to varioustechniques for elucidation of the DNA sequence. In this way, mutationsin the DNA sequence may be detected and used to diagnose infection andto serotype or classify the infectious agent.

The invention provides a process for diagnosing, disease, preferablybacterial infections, more preferably infections by Staphylococcusaureus, and most preferably disease, such as, infections of the upperrespiratory tract (e.g., otitis media, bacterial tracheitis, acuteepiglottitis, thyroiditis), lower respiratory (e.g., empyema, lungabscess), cardiac (e.g., infective endocarditis), gastrointestinal(e.g., secretory diarrhoea, splenic absces, retroperitoneal abscess),CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis,keratitis, endophthalmitis, preseptal and orbital cellulitis,darcryocystitis), kidney and urinary tract (e.g., epididymitis,intrarenal and perinephric absces, toxic shock syndrome), skin (e.g.,impetigo, folliculitis, cutaneous abscesses, cellulitis, woundinfection, bacterial myositis) bone and joint (e.g., septic arthritis,osteomyelitis), comprising determining from a sample derived from anindividual a increased level of expression of polynucleotide having thesequence of FIG. 1 SEQ ID NO: 1! or FIG. 3 SEQ ID NO: 3!. Increased ordecreased expression of tRNA synthetase polynucleotide can be measuredusing any on of the methods well known in the art for the quantation ofpolynucleotides, such as, for example, amplification, PCR, RT-PCR, RNaseprotection, Northern blotting and other hybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of tRNA synthetase protein compared to normalcontrol tissue samples may be used to detect the presence of aninfection, for example. Assay techniques that can be used to determinelevels of a tRNA synthetase protein, in a sample derived from a host arewell-known known to those of skill in the art. Such assay methodsinclude radioimmunoassays, competitive-binding assays, Western Blotanalysis and ELISA assays.

Antibodies

The polypeptides of the invention or variants thereof, or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. "Antibodies" as used hereinincludes monoclonal and polyclonal antibodies, chimeric, single chain,simianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunolglobulin expressionlibrary.

Antibodies generated against the polypeptides of the invention can beobtained by administering the polypeptides or epitope-bearing fragments,analogues or cells to an animal, preferably a nonhuman, using routineprotocols. For preparation of monoclonal antibodies, any technique knownin the art which provides antibodies produced by continuous cell linecultures can be used. Examples include various techniques, such as thosein Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor etal., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies.

Alternatively phage display technology could be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-Fbp or from naive libraries (McCafferty, J.et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992)Biotechnology 10, 779-783). The affinity of these antibodies can also beimproved by chain shuffling (Clackson, T. et al., (1991) Nature 352,624-628).

If two antigen binding domains are present each domain may be directedagainst a different epitope--termed `bispecific` antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides to purify the polypeptides byaffinity chromatography.

Thus, among others, antibodies against tRNA synthetase may be employedto treat infections, particularly bacterial infections and especiallydisease, such as, infections of the upper respiratory tract (e.g.,otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis),lower respiratory (e.g., empyema lung abscess), cardiac (e.g., infectiveendocarditis), gastrointestinal (e.g., secretory diarrhoea, splenicabsces, retroperitoneal abscess), CNS (e.g., cerebral abscess), eye(e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis,preseptal and orbital cellulitis, darcryocystitis), kidney and urinarytract (e.g., epididymitis, intrarenal and perinephric absces, toxicshock syndrome), skin (e.g., impetigo, folliculitis, cutaneousabscesses, cellulitis, wound infection, bacterial myositis) bone andjoint (e.g., septic arthritis, osteomyelitis).

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants which form a particular aspect ofthis invention. The term "antigenically equivalent derivative" as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognised by certain antibodies which, when raised to theprotein or polypeptide according to the invention, interfere with theimmediate physical interaction between pathogen and mammalian host. Theterm "immunologically equivalent derivative" as used herein encompassesa peptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe immediate physical interaction between pathogen and mammalian host.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof is used as an antigen to immunizea mouse or other animal such as a rat or chicken. The fusion protein mayprovide stability to the polypeptide. The antigen may be associated, forexample by conjugation, with an immunogenic carrier protein for examplebovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Alternatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be "humanized"; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal.,(1991) Biotechnology 9, 266-273.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992,1: 363, Manthorpe et al., Hum. Gene Ther. 1963: 4, 419), delivery of DNAcomplexed with specific protein carriers (Wu et al., J Biol Chem 1989:264,16985), coprecipitation of DNA with calcium phosphate (Benvenisty &Reshef, PNAS, 1986: 83,9551), encapsulation of DNA in various forms ofliposomes (Kaneda et al., Science 1989: 243,375), particle bombardment(Tang et al., Nature 1992, 356: 152. Eisenbraun et al., DNA Cell Biol1993, 12: 791) and in vivo infection using cloned retroviral vectors(Seeger et al., PNAS 1984: 81,5849).

Polypeptides of the invention may also be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics. See, e.g. Coligan et al., CurrentProtocols in Immunology 1(2): Chapter 5 (1991).

Antagonists and agonists--assays and molecules

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action of tRNAsynthetase polypeptides or polynucleotides.

For example, to screen for agonists or antagoists, a synthetic reactionmix, a cellular compartment, such as a membrane, cell envelope or cellwall, or a preparation of any thereof, comprising tRNA synthetasepolypeptide and a labeled substrate of such polypeptide is incubated inthe absence or the presence of a candidate molecule which may be a tRNAsynthetase agonist or antagonist. The ability of the candidate moleculeto agonize or antagonize the tRNA synthetase polypeptide is reflected indecreased binding of the labeled ligand or decreased production ofproduct from such substrate. Molecules which bind gratuitously, i e.,without inducing the effects of tRNA synthetase are most likely to begood antagonists. Molecules that bind well and increase the rate ofproduct production from substrate are agonists. The rate or level ofproduction of product from substrate may be enhanced by using a reportersystem. Reporter systems that may be useful in this regard include butare not limited to colorimetric labeled substrate converted intoproduct, a reporter gene that is responsive to changes in tRNAsynthetase activity, and binding assays known in the art.

Another example of an assay for tRNA synthetase antagonists is acompetitive assay that combines tRNA synthetase and a potentialantagonist with tRNA synthetase-binding molecules, recombinant tRNAsynthetase binding molecules, natural substrates or ligands, orsubstrate or ligand mimetics, under appropriate conditions for acompetitive inhibition assay. tRNA synthetase can be labeled, such as byradioactivity or a colorimetric compound, such that the number of tRNAsynthetase molecules bound to a binding molecule or converted to productcan be determined accurately to assess the effectiveness of thepotential antagonist.

In a further aspect, this invention provides a method of screening drugsto identify those which interfere with the interaction of the novel tRNAsynthetase. The enzyme mediated incorporation of radiolabelled aminoacid into tRNA may be measured by the aminoacylation method whichmeasures amino acid-tRNA as trichloroacetic acid-precipitableradioactivity from radiolabelled amino acid in the presence of tRNA andATP (Hughes J, Mellows G and Soughton S, 1980, FEBS Letters, 122:322-324). Thus inhibitors of novel tRNA synthetase can be detected by areduction in the trichloroacetic acid precipitable radioactivityrelative to the control. Alternatively, novel tRNA synthetase catalysedpartial PPi/ATP exchange reaction which measures the formation ofradiolabelled ATP from PPi can be used to detect novel tRNA synthetaseinhibitors (Calender R & Berg P, 1966, Biochemistry, 5, 1681-1690).

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polypeptide of the inventionand thereby inhibit or extinguish its activity. Potential antagonistsalso may be small organic molecules, a peptide, a polypeptide such as aclosely related protein or antibody that binds the same sites on abinding molecule, such as a binding molecule, without inducing tRNAsynthetase-induced activities, thereby preventing the action of tRNAsynthetase by excluding tRNA synthetase from binding.

Potential antagonists include a small molecule which binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of tRNA synthetase.

In a particular aspect the invention provides the use of thepolypeptide, polynucleotide or inhibitor of the invention to interferewith the initial physical interaction between a pathogen and mammalianhost responsible for sequelae of infection. In particular the moleculesof the invention may be used: i) in the prevention of adhesion ofbacteria, in particular gram positive bacteria, to mammalianextracellular matrix proteins on in-dwelling devices or to extracellularmatrix proteins in wounds; ii) to block tRNA synthetase protein mediatedmammalian cell invasion by, for example, initiating phosphorylation ofmammalian tyrosine kinases (Rosenshine et al., Infect. Immun. 60: 2211(1992); iii) to block bacterial adhesion between mammalian extracellularmatrix proteins and bacterial tRNA synthetase proteins which mediatetissue damage; iv) to block the normal progression of pathogenesis ininfections initiated other than by the implantation of in-dwellingdevices or by other surgical techniques.

Each of the DNA sequences provided herein may be used in the discoveryand development of antibacterial compounds. The encoded protein uponexpression can be used as a target for the screening of antibacterialdrugs. Additionally, the DNA sequences encoding the amino terminalregions of the encoded protein or Shine-Delgarno or other translationfacilitating sequences of the respective mRNA can be used to constructantisense sequences to control the expression of the coding sequence ofinterest.

The antagonists and agonists may be employed for instance to inhibitdisease, such as, infections of the upper respiratory tract (e.g.,otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis),lower respiratory (e.g., empyema, lung abscess), cardiac (e.g.,infective endocarditis), gastrointestinal (e.g., secretory diarrhoea,splenic absces, retroperitoneal abscess), CNS (e.g., cerebral abscess),eye (e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis,preseptal and orbital cellulitis, darcryocystitis), kidney and urinarytract (e.g., epididymitis, intrarenal and perinephric absces, toxicshock syndrome), skin (e.g., impetigo, folliculitis, cutaneousabscesses, cellulitis, wound infection, bacterial myositis) bone andjoint (e.g., septic arthritis, osteomyelitis).

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with tRNA synthetase, or a fragmentor variant thereof, adequate to produce antibody to protect saidindividual from infection, particularly bacterial infection and mostparticularly Staphylococcus aureus infections. Yet another aspect of theinvention relates to a method of inducing immunological response in anindividual which comprises, through gene therapy, delivering geneencoding tRNA synthetase, or a fragment or a variant thereof, forexpressing tRNA synthetase, or a fragment or a variant thereof in vivoin order to induce an immunological response to produce antibody toprotect said individual from disease.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into a host capable or having inducedwithin it an immunological response, induces an immunological responsein such host to a tRNA synthetase or protein coded therefrom, whereinthe composition comprises a recombinant tRNA synthetase or protein codedtherefrom comprising DNA which codes for and expresses an antigen ofsaid tRNA synthetase or protein coded therefrom.

The tRNA synthetase or a fragment thereof may be fused with co-proteinwhich may not by itself produce antibodies, but is capable ofstabilizing the first protein and producing a fused protein which willhave immunogenic and protective properties. Thus fused recombinantprotein, preferably further comprises an antigenic co-protein, such asGlutathione-S-transferase (GST) or beta-galactosidase, relatively largeco-proteins which solubilise the protein and facilitate production andpurification thereof. Moreover, the co-protein may act as an adjuvant inthe sense of providing a generalized stimulation of the immune system.The co-protein may be attached to either the amino or carboxy terminusof the first protein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Staphylococcus aureus will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of bacterial infection, particularlyStaphylococcus aureus infections, in mammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host toproduce specific antibodies which protect against invasion of bacteria,for example by blocking adherence of bacteria to damaged tissue.Examples of tissue damage include wounds in skin or connective tissuecaused e.g. by mechanical, chemical or thermal damage or by implantationof indwelling devices, or wounds in the mucous membranes, such as themouth, mammary glands, urethra or vagina.

The invention also includes a vaccine formulation which comprises theimmunogenic recombinant protein together with a suitable carrier. Sincethe protein may be broken down in the stomach, it is preferablyadministered parenterally, including, for example, administration thatis subcutaneous, intramuscular, intravenous, or intradermal.Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation instonicwith the bodily fluid, preferably the blood, of the individual; andaqueous and non-aqueous sterile suspensions which may include suspendingagents or thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example, sealed ampoules andvials and may be stored in a freeze-dried condition requiring only theaddition of the sterile liquid carrier immediately prior to use. Thevaccine formulation may also include adjuvant systems for enhancing theimmunogenicity of the formulation, such as oil-in water systems andother systems known in the art. The dosage will depend on the specificactivity of the vaccine and can be readily determined by routineexperimentation.

While the invention has been described with reference to certain tRNAsynthetase, it is to be understood that this covers fragments of thenaturally occurring protein and similar proteins with additions,deletions or substitutions which do not substantially affect theimmunogenic properties of the recombinant protein.

Compositions, kits and administration

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or the agonists or antagonists. Thepolypeptides of the invention may be employed in combination with anon-sterile or sterile carrier or carriers for use with cells, tissuesor organisms, such as a pharmaceutical carrier suitable foradministration to a subject. Such compositions comprise, for instance, amedia additive or a therapeutically effective amount of a polypeptide ofthe invention and a pharmaceutically acceptable carrier or excipient.Such carriers may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol and combinations thereof. Theformulation should suit the mode of administration. The inventionfurther relates to diagnostic and pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the invention may be employed aloneor in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters,etc.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStaphylococcus aureus wound infections.

Many orthopaedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteremia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 μg/kg of antigen, and such dose ispreferably administered 1-3 times and with an interval of 1-3 weeks.With the indicated dose range, no adverse toxicological effects will beobserved with the compounds of the invention which would preclude theiradministration to suitable individuals.

The antibodies described above may also be used as diagnostic reagentsto detect the presence of bacteria containing the tRNA synthetaseprotein.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1 Library Production

The polynucleotide having the DNA sequence given in SEQ ID NO: 1 wasobtained from a library of clones of chromosomal DNA of Staphylococcusaureus in E. coli. In some cases the sequencing data from two or moreclones containing overlapping Staphylococcus aureus DNAs was used toconstruct the contiguous DNA sequence in SEQ ID NO: 1. Libraries may beprepared by routine methods, for example:

Methods 1 and 2 below.

Total cellular DNA is isolated from Staphylococcus aureus WCUH 29according to standard procedures and size-fractionated by either of twomethods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E.coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bshl2351), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E.coli infected with the packagedlibrary. The library is amplified by standard procedures.

Example 2 Measurement of Phenylalanyl tRNA Synthetase (FRS) Activity

The enzyme catalyses the aminoacylation of tRNA^(Phe), which proceedsthrough a two step mechanism. The first step involves the formation of astable enzyme:phenylalanyl adenylate complex resulting from the specificbinding and reaction of ATP and L-phenylalanine. Subsequently, the 3'terminal adenosine of enzyme-bound tRNAPhe reacts with theaminoacyladenylate, leading to the esterification of the tRNA andrelease of AMP. These steps are summarised below; ##EQU2##

This reaction can be assayed in order to characterise the enzyme oridentify specific inhibitors of its activity in a number of ways:

1. Measurement of the formation of Phe-tRNA^(Phe) can be specificallydetermined using radiolabelled phenylalanine and separating freephenylalanine from Phe-tRNA using precipitation/filtration techniques(e.g. in cold trichloroacetic acid¹,2)

2. The full acylation reaction can also be measured by analysingproduction of either PPi or AMP which are produced in stoichiometricratio to the tRNA acylation. This may be achieved in a number of ways,for example using colorimetric³ ; or enzyme coupled⁴ measurement of Piafter addition of excess inorganic pyrophosphatase or using enzymecoupled assays to directly measure AMP or PPi production⁵.

3. The partial reaction (a) can be assayed through radiolabel isotopicexchange between ATP and PPi, since each of the steps in this part ofthe reaction are freely reversible. This reaction is typically has ak_(cat) around 20-fold higher than the full acylation reaction (a+b),and is readily measured using chromatographic principles which separatePPi from ATP (i.e. using activated charcoal¹,2.

Ligand Binding to FRS.

It is also possible to define ligand interactions with FRS inexperiments that are not dependent upon enzyme catalysed turnover ofsubstrates. This type of experiment can be done in a number of ways:

1. Effects of ligand binding upon enzyme intrinsic fluorescence (e.g. oftryptophan). Binding of either natural ligands or inhibitors may resultin enzyme conformational changes which alter enzyme fluorescence. Usingstopped-flow fluorescence equipment, this can be used to define themicroscopic rate constants that describe binding. Alternatively,steady-state fluorescence titration methods can yield the overalldissociation constant for binding in the same way that these areaccessed through enzyme inhibition experiments

2. Spectral effects of ligands. Where the ligands themselves are eitherfluorescent or possess chromophores that overlap with enzyme tryptophanfluorescence, binding can be detected either via changes in the ligandfluorescence properties (e.g. intensity, lifetime or polarisation) orfluorescence resonance energy transfer with enzyme tryptophans. Theligands could either be inhibitors or variants of the natural ligands(i.e. fluorescent ATP derivatives or tRNAPhe labelled with afluorophore).

3. Thermal analysis of the enzyme:ligand complex. Using calorimetrictechniques (e.g. Isothermal Calorimetry, Differential ScanningCalorimetry) it is possible to detect thermal changes, or shifts in thestability of FRS which reports and therefore allows the characterisationof ligand binding.

References

1. Calender & Berg (1966) Biochemistry 5, 1681-1690

2. Toth MJ & Schimmel P (1990) J. Biol. Chem. 265, 1000-1004

3. Hoenig (1989) J. Biochem. Biophys. Meth. 19, 249-252

4. Webb TM (1994) Anal. Biochem. 218,449-454

5. Sigma Chemicals Catalogue, 1986

Example 3 Aminoacylation Assays for FRS Activity

Assays are performed either using purified S. aureus FRS overexpressedin E. coli, or using crude cell lysate from E. coli overexpressing FRS.The latter usually contains around 10% of total protein as FRS. Enzymeis stored at -70° C. in 50 mM Tris-HCl buffer (pH 7.8), 10 mM MgCl₂ and10 mM B-mercaptoethanol after flash freezing in liquid N₂. Inexperiments to determine the activity of enzyme samples, these stocksare diluted over a wide range (100 fold to 10,000 fold) in 50 mM Tris pH7.8, 10 mM MgCl2, 1 mM Dithiothreitol and stored on ice prior to assay.

The assay procedure is as follows; 50 ml of enzyme prepared and dilutedas described above is mixed with reaction mixture (100 ml), comprising:0.25 uCi L- U-¹⁴ C!-Phenylalanine (Amersham International), 4 mg/ml E.coli MRE600 mixed tRNA (from Boehringer Manheim), 5 mM ATP, 15 mM MgSO₄,3 mM DTT, 75 mM KCl and 50 mM Tris-HCI pH 7.8. Unless otherwise stated,all reagents are obtained from Sigma Chemical Company Ltd.Concentrations are given as in the final reaction mix. After addition ofthe enzyme to start the reaction, assay samples are incubated at 37° C.and, at the desired time, duplicate aliquots (50 ul) are removed andquenched with 7% trichloroacetic acid (100 ul) and left on ice for 30min. The precipitates are harvested using a Packard Filtermate 196 CellHarvester Packard Instruments Ltd.! onto glass fibre filters which arewashed successively with 7% trichloroacetic acid and ethanol. Thefilters are dried at 70° C. for 1 hour and the levels of radioactivitymeasured by scintillation counting (Packard Topcount).

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 4                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1056 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Genomic DNA                                         -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - ATGTCTGAAC AACAAACAAT GTCAGAGTTA AAACAACAAG CGCTTGTAGA TA - #TTAATGAA         60                                                                          - GCAAATGATG AACGTGCACT GCAAGAAGTT AAAGTGAAAT ACTTAGGTAA AA - #AAGGGTCA        120                                                                          - GTTAGCGGAC TAATGAAATT GATGAAGGAT TTGCCGAATG AAGAGAAACC TG - #CGTTTGGT        180                                                                          - CAAAAAGTGA ATGAATTGCG TCAAACAATT CAAAATGAAT TAGATGAAAG AC - #AACAGATG        240                                                                          - TTAGTTAAAG AAAAATTAAA TAAGCAATTG GCTGAAGAAA CAATTGATGT AT - #CATTACCA        300                                                                          - GGTCGTCATA TTGAAATCGG TTCAAAGCAT CCATTAACAC GTACAATAGA AG - #AAATTGAA        360                                                                          - GACTTATTCT TAGGTTTAGG TTATGAAATT GTGAATGGAT ATGAAGTTGA AC - #AAGATCAT        420                                                                          - TATAACTTCG AAATGCTGAA TTTACCTAAA TCACACCCTG CACGTGATAT GC - #AAGATAGT        480                                                                          - TTCTATATTA CGGATGAAAT TTTATTACGT ACGCATACAT CACCAGTGCA GG - #CACGTACG        540                                                                          - ATGGAATCAC GTCATGGTCA AGGTCCAGTT AAAATTATTT GCCCTGGTAA AG - #TGTATCGT        600                                                                          - CGTGACTCTG ATGATGCGAC ACATAGTCAT CAATTTACAC AAATCGAAGG AT - #TAGTTGTT        660                                                                          - GATAAAAACG TTAAAATGAG TGATTTGAAA GGCACTTTAG AATTGTTAGC TA - #AGAAATTA        720                                                                          - TTTGGTGCTG ATCGTGAAAT TCGTTTACGT CCAAGTTACT TCCCATTCAC TG - #AACCTTCT        780                                                                          - GTAGAAGTTG ATGTGTCATG TTTTAAATGT AAAGGAAAAG GTTGTAATGT GT - #GTAAACAC        840                                                                          - ACAGGATGGA TTGAAATTTT AGGTGCTGGA ATGGTACATC CTAATGTATT AG - #AAATGGCT        900                                                                          - GGTTTTGATT CTTCAGAGTA CTCTGGATTT GCATTTGGTA TGGGACCAGA CC - #GTATTGCA        960                                                                          - ATGTTGAAAT ATGGTATAGA AGATATTCGT CATTTCTATA CTAATGATGT GA - #GATTTTTA       1020                                                                          #     1056         TAGA AGATAGAGGT GACATG                                     - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 352 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Ser Glu Gln Gln Thr Met Ser Glu Leu Ly - #s Gln Gln Ala Leu Val         #                15                                                           - Asp Ile Asn Glu Ala Asn Asp Glu Arg Ala Le - #u Gln Glu Val Lys Val         #            30                                                               - Lys Tyr Leu Gly Lys Lys Gly Ser Val Ser Gl - #y Leu Met Lys Leu Met         #        45                                                                   - Lys Asp Leu Pro Asn Glu Glu Lys Pro Ala Ph - #e Gly Gln Lys Val Asn         #    60                                                                       - Glu Leu Arg Gln Thr Ile Gln Asn Glu Leu As - #p Glu Arg Gln Gln Met         #80                                                                           - Leu Val Lys Glu Lys Leu Asn Lys Gln Leu Al - #a Glu Glu Thr Ile Asp         #                95                                                           - Val Ser Leu Pro Gly Arg His Ile Glu Ile Gl - #y Ser Lys His Pro Leu         #           110                                                               - Thr Arg Thr Ile Glu Glu Ile Glu Asp Leu Ph - #e Leu Gly Leu Gly Tyr         #       125                                                                   - Glu Ile Val Asn Gly Tyr Glu Val Glu Gln As - #p His Tyr Asn Phe Glu         #   140                                                                       - Met Leu Asn Leu Pro Lys Ser His Pro Ala Ar - #g Asp Met Gln Asp Ser         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Phe Tyr Ile Thr Asp Glu Ile Leu Leu Arg Th - #r His Thr Ser Pro Val         #               175                                                           - Gln Ala Arg Thr Met Glu Ser Arg His Gly Gl - #n Gly Pro Val Lys Ile         #           190                                                               - Ile Cys Pro Gly Lys Val Tyr Arg Arg Asp Se - #r Asp Asp Ala Thr His         #       205                                                                   - Ser His Gln Phe Thr Gln Ile Glu Gly Leu Va - #l Val Asp Lys Asn Val         #   220                                                                       - Lys Met Ser Asp Leu Lys Gly Thr Leu Glu Le - #u Leu Ala Lys Lys Leu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Phe Gly Ala Asp Arg Glu Ile Arg Leu Arg Pr - #o Ser Tyr Phe Pro Phe         #               255                                                           - Thr Glu Pro Ser Val Glu Val Asp Val Ser Cy - #s Phe Lys Cys Lys Gly         #           270                                                               - Lys Gly Cys Asn Val Cys Lys His Thr Gly Tr - #p Ile Glu Ile Leu Gly         #       285                                                                   - Ala Gly Met Val His Pro Asn Val Leu Glu Me - #t Ala Gly Phe Asp Ser         #   300                                                                       - Ser Glu Tyr Ser Gly Phe Ala Phe Gly Met Gl - #y Pro Asp Arg Ile Ala         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Met Leu Lys Tyr Gly Ile Glu Asp Ile Arg Hi - #s Phe Tyr Thr Asn Asp         #               335                                                           - Val Arg Phe Leu Asp Gln Phe Lys Ala Val Gl - #u Asp Arg Gly Asp Met         #           350                                                               - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2400 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: Genomic DNA                                         -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - ATGTTGATAT CAAATGAATG GTTGAAAGAA TATGTAACAA TCGATGATTC TG - #TAAGTGAT         60                                                                          - TTGGCAGAAC GTATTACGCG CACAGGTATT GAAGTGGATG ATTTAATTGA CT - #ACACAAAA        120                                                                          - GATATCAAAA ATTTAGTTGT CGGTTTCGTT AAGTCAAAAG AGAAACATCC TG - #ATGCCGAT        180                                                                          - AAATTAAATG TTTGCCAAGT TGATATCGGA GAAGACGAAC CTGTACAAAT CG - #TATGTGGT        240                                                                          - GCACCGAACG TTGATGCAGG ACAATATGTC ATTGTTGCTA AAGTAGGTGG CA - #GATTGCCT        300                                                                          - GGTGGTATTA AAATTAAGCG TGCCAAATTA CGCGGTGAAC GTTCAGAAGG TA - #TGATTTGT        360                                                                          - TCGTTACAAG AAATTGGTAT TTCAAGTAAC TATATACCGA AAAGTTTTGA AT - #CAGGCATT        420                                                                          - TATGTTTTTA GTGAATCCCA AGTTCCAGGA ACAGATGCCT TACAAGCTTT AT - #ATTTAGAT        480                                                                          - GATCAAGTAA TGGAATTTGA TTTAACGCCG AATCGTGCAG ATGCTTTAAG TA - #TGATAGGT        540                                                                          - ACTGCTTATG AAGTTGCAGC ATTATATAAT ACAAAAATGA CTAAGCCAGA GA - #CAACATCA        600                                                                          - AATGAGCTTG AGTTATCTGC AAATGATGAA TTGACTGTGA CAATTGAAAA TG - #AAGATAAA        660                                                                          - GTACCATATT ATAGTGCACG TGTTGTTCAC GACGTGACAA TTGAACCTTC GC - #CAATTTGG        720                                                                          - ATGCAAGCAC GCTTAATAAA AGCGGGTATA CGTCCTATTA ATAATGTTGT TG - #ACATTTCA        780                                                                          - AATTATGTGT TATTAGAATA CGGTCAACCA TTGCACATGT TTGATCAAGA TG - #CGATTGGT        840                                                                          - TCACAACAAA TTGTTGTTCG TCAAGCTAAT GAAGGCGAAA AAATGACAAC AT - #TAGATGAT        900                                                                          - ACAGAACGTG AATTATTAAC GAGCGATATT GTCATTACTA ATGGACAAAC TC - #CAATTGCA        960                                                                          - TTAGCTGGTG TTATGGGTGG CGATTTTTCA GAAGTTAAAG AACAAACATC AA - #ATATAGTG       1020                                                                          - ATTGAAGGTG CTATTTTTGA TCCAGTTTCA ATTCGTCATA CATCAAGACG TT - #TAAATTTA       1080                                                                          - CGCAGTGAAT CATCTAGTCG TTTTGAAAAA GGAATAGCTA CTGAATTTGT AG - #ATGAAGCA       1140                                                                          - GTCGACCGTG CATGTTATTT ATTACAAACT TATGCAAACG GAAAAGTGCT AA - #AAGATAGA       1200                                                                          - GTGTCTTCAG GAGAACTTGG TGCATTTATT ACACCAATCG ACATCACTGC TG - #ATAAAATT       1260                                                                          - AATCGCACTA TTGGATTTGA TTTGTCACAA AATGATATTG TTACTATTTT TA - #ATCAACTA       1320                                                                          - GGGTTTGATA CAGAAATAAA TGATGATGTT ATTACAGTGC TAGTACCATC AC - #GTCGTAAA       1380                                                                          - GATATTACAA TTAAAGAAGA TTTAATTGAA GAAGTTGCAC GTATATATGG AT - #ACGACGAT       1440                                                                          - ATTCCATCAA CGTTACCTGT CTTCGATAAA GTTACTAGTG GTCAGCTAAC TG - #ATCGCCAA       1500                                                                          - TATAAAACTA GAATGGTTAA AGAAGTGTTA GAAGGTGCTG GATTAGATCA AG - #CTATTACG       1560                                                                          - TATTCGTTAG TTTCTAAAGA GGATGCTACT GCATTTTCGA TGCAACAGCG TC - #AAACAATT       1620                                                                          - GATTTATTGA TGCCAATGAG TGAAGCGCAT GCGTCATTAC GTCAAAGTTT AT - #TACCACAT       1680                                                                          - TTAATCGAAG TAGCATCATA TAATGTGGCA CGCAAAAATA AAGATGTAAA AT - #TATTTGAA       1740                                                                          - ATCGGCAATG TCTTCTTTGC TAATGGAGAA GGTGAACTAC CAGATCAAGT TG - #AATATTTA       1800                                                                          - AGTGGTATTT TAACTGGAGA TTATGTAGTC AATCAATGGC AAGGTAAGAA AG - #AAACGGTT       1860                                                                          - GATTTCTATT TAGCAAAAGG TGTCGTGGAT CGAGTATCTG AAAAGTTAAA CC - #TTGAATTT       1920                                                                          - AGTTATCGTC GTGCTGATAT TGATGGATTA CATCCAGGTC GTACTGCTGA AA - #TCTTATTA       1980                                                                          - GAGAATAAAG TTGTTGGTTT TATTGGTGAA TTACATCCAA CATTAGCAGC TG - #ATAATGAT       2040                                                                          - TTAAAACGTA CGTATGTTTT TGAGTTGAAT TTTGATGCAT TAATGGCTGT GT - #CGGTAGGT       2100                                                                          - TACATTAATT ACCAGCCAAT TCCGAGATTC CCAGGCATGT CTCGTGACAT TG - #CATTAGAA       2160                                                                          - GTAGATCAAA ATATTCCAGC AGCTGATTTA TTATCAACGA TTCATGCACA CG - #GTGGCAAT       2220                                                                          - ATATTAAAAG ATACACTTGT CTTTGATGTA TATCAGGGCG AACATTTAGA AA - #AAGGTAAA       2280                                                                          - AAATCAATTG CAATACGTTT AAATTATTTA GACACAGAAG AAACATTGAC AG - #ATGAGCGC       2340                                                                          - GTTTCAAAAG TGCAAGCGGA AATTGAAGCA GCATTAATTG AACAAGGTGC TG - #TTATTAGA       2400                                                                          - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 800 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Met Leu Ile Ser Asn Glu Trp Leu Lys Glu Ty - #r Val Thr Ile Asp Asp         #                15                                                           - Ser Val Ser Asp Leu Ala Glu Arg Ile Thr Ar - #g Thr Gly Ile Glu Val         #            30                                                               - Asp Asp Leu Ile Asp Tyr Thr Lys Asp Ile Ly - #s Asn Leu Val Val Gly         #        45                                                                   - Phe Val Lys Ser Lys Glu Lys His Pro Asp Al - #a Asp Lys Leu Asn Val         #    60                                                                       - Cys Gln Val Asp Ile Gly Glu Asp Glu Pro Va - #l Gln Ile Val Cys Gly         #80                                                                           - Ala Pro Asn Val Asp Ala Gly Gln Tyr Val Il - #e Val Ala Lys Val Gly         #                95                                                           - Gly Arg Leu Pro Gly Gly Ile Lys Ile Lys Ar - #g Ala Lys Leu Arg Gly         #           110                                                               - Glu Arg Ser Glu Gly Met Ile Cys Ser Leu Gl - #n Glu Ile Gly Ile Ser         #       125                                                                   - Ser Asn Tyr Ile Pro Lys Ser Phe Glu Ser Gl - #y Ile Tyr Val Phe Ser         #   140                                                                       - Glu Ser Gln Val Pro Gly Thr Asp Ala Leu Gl - #n Ala Leu Tyr Leu Asp         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Asp Gln Val Met Glu Phe Asp Leu Thr Pro As - #n Arg Ala Asp Ala Leu         #               175                                                           - Ser Met Ile Gly Thr Ala Tyr Glu Val Ala Al - #a Leu Tyr Asn Thr Lys         #           190                                                               - Met Thr Lys Pro Glu Thr Thr Ser Asn Glu Le - #u Glu Leu Ser Ala Asn         #       205                                                                   - Asp Glu Leu Thr Val Thr Ile Glu Asn Glu As - #p Lys Val Pro Tyr Tyr         #   220                                                                       - Ser Ala Arg Val Val His Asp Val Thr Ile Gl - #u Pro Ser Pro Ile Trp         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Met Gln Ala Arg Leu Ile Lys Ala Gly Ile Ar - #g Pro Ile Asn Asn Val         #               255                                                           - Val Asp Ile Ser Asn Tyr Val Leu Leu Glu Ty - #r Gly Gln Pro Leu His         #           270                                                               - Met Phe Asp Gln Asp Ala Ile Gly Ser Gln Gl - #n Ile Val Val Arg Gln         #       285                                                                   - Ala Asn Glu Gly Glu Lys Met Thr Thr Leu As - #p Asp Thr Glu Arg Glu         #   300                                                                       - Leu Leu Thr Ser Asp Ile Val Ile Thr Asn Gl - #y Gln Thr Pro Ile Ala         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Leu Ala Gly Val Met Gly Gly Asp Phe Ser Gl - #u Val Lys Glu Gln Thr         #               335                                                           - Ser Asn Ile Val Ile Glu Gly Ala Ile Phe As - #p Pro Val Ser Ile Arg         #           350                                                               - His Thr Ser Arg Arg Leu Asn Leu Arg Ser Gl - #u Ser Ser Ser Arg Phe         #       365                                                                   - Glu Lys Gly Ile Ala Thr Glu Phe Val Asp Gl - #u Ala Val Asp Arg Ala         #   380                                                                       - Cys Tyr Leu Leu Gln Thr Tyr Ala Asn Gly Ly - #s Val Leu Lys Asp Arg         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Val Ser Ser Gly Glu Leu Gly Ala Phe Ile Th - #r Pro Ile Asp Ile Thr         #               415                                                           - Ala Asp Lys Ile Asn Arg Thr Ile Gly Phe As - #p Leu Ser Gln Asn Asp         #           430                                                               - Ile Val Thr Ile Phe Asn Gln Leu Gly Phe As - #p Thr Glu Ile Asn Asp         #       445                                                                   - Asp Val Ile Thr Val Leu Val Pro Ser Arg Ar - #g Lys Asp Ile Thr Ile         #   460                                                                       - Lys Glu Asp Leu Ile Glu Glu Val Ala Arg Il - #e Tyr Gly Tyr Asp Asp         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Ile Pro Ser Thr Leu Pro Val Phe Asp Lys Va - #l Thr Ser Gly Gln Leu         #               495                                                           - Thr Asp Arg Gln Tyr Lys Thr Arg Met Val Ly - #s Glu Val Leu Glu Gly         #           510                                                               - Ala Gly Leu Asp Gln Ala Ile Thr Tyr Ser Le - #u Val Ser Lys Glu Asp         #       525                                                                   - Ala Thr Ala Phe Ser Met Gln Gln Arg Gln Th - #r Ile Asp Leu Leu Met         #   540                                                                       - Pro Met Ser Glu Ala His Ala Ser Leu Arg Gl - #n Ser Leu Leu Pro His         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Leu Ile Glu Val Ala Ser Tyr Asn Val Ala Ar - #g Lys Asn Lys Asp Val         #               575                                                           - Lys Leu Phe Glu Ile Gly Asn Val Phe Phe Al - #a Asn Gly Glu Gly Glu         #           590                                                               - Leu Pro Asp Gln Val Glu Tyr Leu Ser Gly Il - #e Leu Thr Gly Asp Tyr         #       605                                                                   - Val Val Asn Gln Trp Gln Gly Lys Lys Glu Th - #r Val Asp Phe Tyr Leu         #   620                                                                       - Ala Lys Gly Val Val Asp Arg Val Ser Glu Ly - #s Leu Asn Leu Glu Phe         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Ser Tyr Arg Arg Ala Asp Ile Asp Gly Leu Hi - #s Pro Gly Arg Thr Ala         #               655                                                           - Glu Ile Leu Leu Glu Asn Lys Val Val Gly Ph - #e Ile Gly Glu Leu His         #           670                                                               - Pro Thr Leu Ala Ala Asp Asn Asp Leu Lys Ar - #g Thr Tyr Val Phe Glu         #       685                                                                   - Leu Asn Phe Asp Ala Leu Met Ala Val Ser Va - #l Gly Tyr Ile Asn Tyr         #   700                                                                       - Gln Pro Ile Pro Arg Phe Pro Gly Met Ser Ar - #g Asp Ile Ala Leu Glu         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Val Asp Gln Asn Ile Pro Ala Ala Asp Leu Le - #u Ser Thr Ile His Ala         #               735                                                           - His Gly Gly Asn Ile Leu Lys Asp Thr Leu Va - #l Phe Asp Val Tyr Gln         #           750                                                               - Gly Glu His Leu Glu Lys Gly Lys Lys Ser Il - #e Ala Ile Arg Leu Asn         #       765                                                                   - Tyr Leu Asp Thr Glu Glu Thr Leu Thr Asp Gl - #u Arg Val Ser Lys Val         #   780                                                                       - Gln Ala Glu Ile Glu Ala Ala Leu Ile Glu Gl - #n Gly Ala Val Ile Arg         785                 7 - #90                 7 - #95                 8 -       #00                                                                           __________________________________________________________________________

What is claimed is:
 1. An isolated polypeptide comprising an amino acidsequence as set forth in SEQ ID NO:
 2. 2. An isolated polypeptidecomprising a polypeptide sequence selected from the group consistingof:(a) a first sequence which is SEQ ID NO: 2, (b) a second sequencecomprising a truncation of the first sequence containing at least 30amino acids, (c) a third sequence comprising a truncation of the firstsequence containing at least 50 amino acids, (d) a fourth sequence whichis identical to the first sequence except that the fourth sequence has1-5 mutations relative to the first sequence, wherein each mutation is asubstitution, deletion or insertion of one amino acid, or (e) a fifthsequence which is identical to the first sequence except that the fifthsequence has 5-10 mutations relative to the first sequence, wherein eachmutation is a substitution, deletion or insertion of one amino acid,wherein the isolated polypeptide has tRNA synthetase enzymatic activity.3. The isolated polypeptide of claim 2, wherein the polypeptide sequenceconsists of he first sequence which is SEQ ID NO:
 2. 4. The isolatedpolypeptide of claim 2, wherein the polypeptide sequence consists of thesecond sequence comprising a truncation of the first sequence containingat least 30 amino acids.
 5. The isolated polypeptide of claim 2, whereinthe polypeptide sequence consists of the third sequence comprising atruncation of the first sequence containing at least 50 amino acids. 6.The isolated polypeptide of claim 2, wherein the polypeptide sequenceconsists of the fourth sequence which is identical to the first sequenceexcept that the fourth sequence has 1-5 mutations relative to the firstsequence, wherein each mutation is a substitution, deletion or insertionof one amino acid.
 7. The isolated polypeptide of claim 2, wherein thepolypeptide sequence consists of the fifth sequence which is identicalto the first sequence except that the fifth sequence has 5-10 mutationsrelative to the first sequence, wherein each mutation is a substitution,deletion or insertion of one amino acid.
 8. The isolated polypeptide ofclaim 2, which is encoded by a polynucleotide comprising nucleotides 1to 1056 set forth in SEQ ID NO:
 1. 9. The isolated polypeptide of claim2, wherein said polypeptide is phenylalanyl tRNA synthetase protein. 10.The isolated polypeptide of claim 2, comprising amino acids 1 to 352 ofSEQ ID NO:
 2. 11. An isolated polypeptide encoded by an isolated firstpolynucleotide wherein the isolated first polynucleotide hybridizesunder stringent conditions to a second polylucleotide which encodes themature polypeptide of SEQ ID NO: 2; wherein stringent conditionscomprise overnight incubation at 42° C. in a solution comprising: 50%formamide, 5×SSC (150 mM NacL, 15 mM trisodium citrate), 50 mM sodiumphosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20micrograms/ml denatured, sheared salmon sperm DNA, followed by washingthe filters in 0.1×SSC at about 65° C.; wherein the isolated polypeptideis 1RNA synthetase polypeptide expressed by the DNA contained in NCIMBDeposit No.
 40771. 12. An isolated polypeptide encoded by an isolatedfirst polynucleotide wherein the isolated first polynucleotidehybridizes under stringent conditions to the polynucleotide sequence ofSEQ ID NO: 1, wherein stringent conditions comprise overnight incubationat 42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NacL,15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the filters in 0.1×SSC at about65° C.; wherein the isolated polypeptide comprises a sequence of atleast 30 amino acids.
 13. The isolated polypeptide of claim 12 encodedby an isolated polynucleotide comprising nucleotides 1 to 1056 set forthin SEQ ID NO:
 1. 14. The isolated polypeptide of claim 12 encoded by anisolated polynucleotide comprising the polynucleotide sequence set forthin SEQ ID NO: 1 that encodes a polypeptide of SEQ ID NO: 2 wherein theisolated polypeptide is phenylalanyl tRNA syntherase protein.
 15. Theisolated polypeptide of claim 12 comprising amino acids 1 to 352 of SEQID NO:
 2. 16. An isolated polypeptide comprising a polypeptide sequenceselected from the group consisting of:(a) a first sequence which is SEQID NO: 2, (b) a second sequence comprising a truncation of the firstsequence containing at least 30 amino acids, (c) a third sequencecomprising a truncation of the first sequence containing at least 50amino acids, (d) a fourth sequence which is identical to the firstsequence except that the fourth sequence has 1-5 mutations relative tothe first sequence, wherein each mutation is a substitution, deletion orinsertion of one amino acid, or (e) a fifth sequence which is identicalto the first sequence except that the fifth sequence has 5-10 mutationsrelative to the first sequence, wherein each mutation is a substitution,deletion or insertion of one amino acid, wherein the isolatedpolypeptide is capable of generating antibodies having bindingspecificity for the amino acid sequence set forth in SEQ ID NO:
 2. 17.The isolated polypeptide of claim 16, wherein the polypeptide sequenceconsists of the first sequence which is SEQ ID NO:
 2. 18. The isolatedpolypeptide of claim 16, wherein the polypeptide sequence consists ofthe second sequence comprising a truncation of the first sequencecontaining at least 30 amino acids.
 19. The isolated polypeptide ofclaim 16, wherein the polypeptide sequence consists of the thirdsequence comprising a truncation of the first sequence containing atleast 50 amino acids.
 20. The isolated polypeptide of claim 16, whereinthe polypeptide sequence consists of the fourth sequence which isidentical to the first sequence except that the fourth sequence has 1-5mutations relative to the first sequence, wherein each mutation is asubstitution, deletion or insertion of one amino acid.
 21. The isolatedpolypeptide of claim 16, wherein the polypeptide sequence consists ofthe fifth sequence which is identical to the first sequence except thatthe fifth sequence has 5-10 mutations relative to the first sequence,wherein each mutation is a substitution, deletion or insertion of oneamino acid.
 22. An isolated polypeptide comprising a polypeptidesequence selected from the group consisting of:(a) a first sequencewhich is SEQ ID NO: 4, (b) a second sequence comprising a truncation ofthe first sequence containing at least 30 amino acids, (c) a thirdsequence comprising a truncation of the first sequence containing atleast 50 amino acids, (d) a fourth sequence which is identical to thefirst sequence except that the fourth sequence has 1-5 mutationsrelative to the first sequence, wherein each mutation is a substitution,deletion or insertion of one amino acid, or (e) a fifth sequence whichis identical to the first sequence except that the fifth sequence has5-10 mutations relative to the first sequence, wherein each mutation isa substitution, deletion or insertion of one amino acid, wherein theisolated polypeptide has tRNA synthetase enzymatic activity.
 23. Theisolated polypeptide of claim 22, wherein the polypeptide sequenceconsists of the first sequence which is SEQ ID NO:
 4. 24. The isolatedpolypeptide of claim 22, wherein the polypeptide sequence consists ofthe second sequence comprising a truncation of the first sequencecontaining at least 30 amino acids.
 25. The isolated polypeptide ofclaim 22, wherein the polypeptide sequence consists of the thirdsequence comprising a truncation of the first sequence containing atleast 50 amino acids.
 26. The isolated polypeptide of claim 22, whereinthe polypeptide sequence consists of the fourth sequence which isidentical to the first sequence except that the fourth sequence has 1-5mutations relative to the first sequence, wherein each mutation is asubstitution, deletion or insertion of one amino acid.
 27. The isolatedpolypeptide of claim 22, wherein the polypeptide sequence consists ofthe fifth sequence which is identical to the first sequence except thatthe fifth sequence has 5-10 mutations relative to the first sequence,wherein each mutation is a substitution, deletion or insertion of oneamino acid.
 28. The isolated polypeptide of claim 22, which is encodedby a polynucleotide comprising the polynucleotide sequence set forth inSEQ ID NO:
 3. 29. The isolated polypeptide of claim 22, wherein saidpolypeptide is phenylalanyl tRNA synthetase protein.
 30. The isolatedpolypeptide of claim 2, comprising the amino acid sequence set forth inSEQ ID NO:
 4. 31. An isolated polypeptide encoded by an isolated firstpolynucleotide wherein the isolated first polynucleotide hybridizesunder stringent conditions to a second polynucleotide which encodes themature polypeptide of SEQ ID NO: 4; wherein stringent conditionscomprise overnight incubation at 42° C. in a solution comprising: 50%formamide, 5×SSC (150 mM NaCL, 15 mM trisodium citrate), 50 mM sodiumphosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20micrograms/ml denatured, sheared salmon sperm DNA, followed by washingthe filters in 0.1×SSC at about 65° C.; wherein the isolated polypeptideis tRNA synthetase polypeptide expressed by the DNA contained in NCIMBDeposit No.
 40771. 32. An isolated polypeptide encoded by an isolatedfirst polynucleotide wherein the isolated first polynucleotidehybridizes under stringent conditions to the polynucleotide sequence ofSEQ ID NO: 3, wherein stringent conditions comprise overnight incubationat 42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NacL,15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the filters in 0.1×SSC at about65° C.; wherein the isolated polypeptide comprises a sequence of atleast 30 amino acids.
 33. The isolated polypeptide of claim 32 encodedby an isolated polynucleotide comprising the polynucleotide sequence setforth in SEQ ID NO:
 3. 34. The isolated polypeptide of claim 32 encodedby an isolated polynucleotide comprising the polynucleotide sequence setforth in SEQ ID NO: 3 that encodes a polypeptide of SEQ ID NO: 4 whereinthe isolated polypeptide is phenylalanyl tRNA synthetase protein. 35.The isolated polypeptide of claim 32 comprising the amino acid sequenceset forth in SEQ ID NO:
 4. 36. An isolated polypeptide comprising apolypeptide sequence selected from the group consisting of:(a) a firstsequence which is SEQ ID NO: 4, (b) a second sequence comprising atruncation of the first sequence containing at least 30 amino acids, (c)a third sequence comprising a truncation of the first sequencecontaining at least 50 amino acids, (d) a fourth sequence which isidentical to the first sequence except that the fourth sequence has 1-5mutations relative to the first sequence, wherein each mutation is asubstitution, deletion or insertion of one amino acid, or (e) a fifthsequence which is identical to the first sequence except that the fifthsequence has 5-10 mutations relative to the first sequence, wherein eachmutation is a substitution, deletion or insertion of one amino acid,wherein the isolated polypeptide is capable of generating antibodieshaving binding specificity for the amino acid sequence set forth in SEQID NO:
 4. 37. The isolated polypeptide of claim 36, wherein thepolypeptide sequence consists of the first sequence which is SEQ ID NO:4.
 38. The isolated polypeptide of claim 36, wherein the polypeptidesequence consists of the second sequence comprising a truncation of thefirst sequence containing at least 30 amino acids.
 39. The isolatedpolypeptide of claim 36, wherein the polypeptide sequence consists ofthe third sequence comprising a truncation of the first sequencecontaining at least 50 amino acids.
 40. The isolated polypeptide ofclaim 36, wherein the polypeptide sequence consists of the fourthsequence which is identical to the first sequence except that the fourthsequence has 1-5 mutations relative to the first sequence, wherein eachmutation is a substitution, deletion or insertion of one amino acid. 41.The isolated polypeptide of claim 36, wherein the polypeptide sequenceconsists of the fifth sequence which is identical to the first sequenceexcept that the fifth sequence has 5-10 mutations relative to the firstsequence, wherein each mutation is a substitution, deletion or insertionof one amino acid.
 42. An isolated polypeptide comprising an amino acidsequence as set forth in SEQ ID NO: 4.